Methods for multiplexing amplification reactions

Abstract

A two-step multiplex amplification reaction includes a first step which truncates the standard initial multiplex amplification round to “boost” the sample copy number by only a 100-1000 fold increase in the target. Following the first step the product is divided into optimized secondary single amplification reactions, each containing one of the primer sets that were used previously in the first or multiplexed booster step. The booster step can occur using an aqueous target nucleic acid or using a solid phase archived nucleic acid. In particular, nucleic acid sequences that uniquely identify E. Coli were identified using the multiplex amplification method.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 
     
       1. A method for simultaneously amplifying multiple nucleic acid sequence targets contained in a sample comprising:
 (a) contacting the sample with at least two primer pairs that target different nucleic acid sequences; 
 (b) forming a plurality of first amplification products by pre-amplifying the sample, wherein the pre-amplifying is truncated prior to reaching an amplification reaction plateau; 
 (c) dividing the plurality of first amplification products into at least two distinct amplification reactions; and, 
 (d) amplifying at least one first amplification product within the at least two distinct amplification reactions using at least one primer pair that has been added to the plurality of first amplification products after the pre-amplifying step. 
 
     
     
       2. The method of  claim 1 , wherein the at least two primer pairs include target specific primer pairs. 
     
     
       3. The method of  claim 2 , wherein at least one of the target specific primer pairs is designed to amplify human or bacterial gene targets. 
     
     
       4. The method of  claim 1 , wherein the plurality of first amplification products include nucleic acids from 100 base pairs to 1100 base pairs in length. 
     
     
       5. The method of  claim 1 , wherein the plurality of first amplification products include nucleic acids having from 30% to 70% GC content. 
     
     
       6. The method of  claim 1 , wherein the pre-amplifying in step (b) or the amplifying in step (d) or both the pre-amplifying in step (b) and the amplifying in step (d) includes performing a polymerase chain reaction. 
     
     
       7. The method of  claim 1 , wherein the pre-amplifying in step (b) or the amplifying in step (d) or both the pre-amplifying in step (b) and the amplifying in step (d) includes performing an isothermal amplification reaction. 
     
     
       8. The method of  claim 1 , wherein the amplifying in step (d) includes performing a polymerase chain reaction, a strand displacement amplification or an isothermal amplification. 
     
     
       9. The method of  claim 1 , further comprising detecting at least one of the nucleic acid sequence targets. 
     
     
       10. The method of  claim 1 , wherein at least one of the nucleic acid sequence targets includes genomic DNA. 
     
     
       11. The method of  claim 1 , wherein at least one of the nucleic acid sequence target includes DNA or RNA extracted from tissue or culture samples. 
     
     
       12. The method of  claim 1 , wherein the pre-amplifying is truncated by exhaustion of at least one of the at least two primer pairs prior to reaching the amplification reaction plateau. 
     
     
       13. The method of  claim 1 , wherein the pre-amplifying is truncated by limiting primer concentration to permit only a few logs of amplification. 
     
     
       14. The method of  claim 1 , wherein the plurality of first amplification products are amplified into early logarithmic phase. 
     
     
       15. The method of  claim 1 , wherein the at least two distinct amplification reactions comprise singleplex amplification reactions. 
     
     
       16. The method of  claim 1 , wherein the at least two primer pairs of step (a) are present at a final concentration per primer of 4 nm. 
     
     
       17. A method for simultaneously amplifying a plurality of nucleic acid sequence targets comprising:
 (a) performing a first round of amplification to form a plurality of first amplification products, comprising contacting a plurality of nucleic acid sequence targets with a plurality of pre-amplification primer pairs specific to one or more of the plurality of nucleic acid sequence targets, wherein the first round of amplification is truncated prior to reaching an amplification reaction plateau; 
 (b) dividing the plurality of first amplification products into at least two distinct amplification reactions; and, 
 (c) performing a second round of amplification within at least one of the at least two distinct reactions to form a plurality of second round amplification products, comprising contacting at least one amplification product from the plurality of first amplification products with at least one amplification primer pair, wherein the at least one amplification primer pair is added after the first round of amplification. 
 
     
     
       18. The method of  claim 17 , further comprising detecting at least one of the plurality of second round amplification products. 
     
     
       19. The method of  claim 17 , wherein the first round of amplification or the second round of amplification or both the first round of amplification and the second round of amplification includes performing a polymerase chain reaction. 
     
     
       20. The method of  claim 17 , wherein the first round of amplification or the second round of amplification or both the first round of amplification and the second round of amplification includes performing an isothermal amplification reaction.